Bone loss is associated with a variety of conditions in which increased production of pro-inflammatory cytokines promotes bone loss by increasing osteoclast formation, activation and survival. Most published data indicate that these effects are mediated mainly indirectly through stromal cell production of M-CSF and RANKL, which bind to their receptors on osteoclasts and activate c-Fos/AP-1, NFkappaB, and NFAT2, expression of each of which is required for osteoclast formation. However, osteoclasts express receptors for a growing list of agents, including pro-inflammatory cytokines, suggesting that direct and indirect effects of cytokines on osteoclasts could combine to induce bone loss. Our over-riding hypothesis is that in disease states, osteoclast numbers may be regulated by direct effects of cytokines on cells in the osteoclast lineage that can lead to self perpetuating, up-regulatory cycles within these cells and subsequently to bone loss. However, the precise mechanisms and the signaling molecules involved remain to be fully elucidated. We plan to investigate the direct effects of cytokines on osteoclasts and their precursors and to determine the roles of c-Fos/AP-1, NFkappaB and NFAT2 signaling in this process using NFkappaB dKO, TNF transgenic and wild-type mice. We propose the following Specific Aims: 1. To fully characterize the effects of cytokines and retroviral transfer of c-Fos/AP-1 genes to wt and NFkB dKO splenocytes and to determine if other osteoclast stimulating factors induce osteoclast formation directly when c-Fos is expressed; 2. To determine the mechanisms whereby retroviral transfer of c-Fos and cytokines induce osteoclast formation directly and to examine the role of NFkB in this process; 3. To examine if cytokine and c-Fos/AP-1 expression are up-regulated in osteoclasts and their precursors in models of cytokine-mediated bone disease. These studies will better define the roles of key signaling pathways and advance our understanding of the mechanisms that directly regulate osteoclast formation and survival. Ultimately they should lead to the development of specific therapeutic agents to prevent and treat common bone diseases associated with increased osteoclast activity.